The structure of the hexameric atrazine chlorohydrolase AtzA

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organism Pseudomonas sp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e. atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit

Tissue-specific expression of a human Polymorphic Epithelial mucin (MUCI) in transgenic mice

The human MUC1 gene codes for the core protein of a mucin which is expressed by glandular epithelia and the carcinomas which develop from these tissues. The core protein is aberrantly glycosylated in cancers, and some antibodies show specificity in their reactions with the cancer-associated mucin, which also contains epitopes recognized by T-cells from breast and pancreatic cancer patients. For evaluating the potential use of mucin-reactive antibodies and mucin-based immunogens in cancer patients, a mouse model, expressing the MUC1 gene product PEM (polymorphic epithelial mucin) as a self antigen, would be extremely useful. To this end, we have developed transgenic mouse strains expressing the human MUC1 gene product in a tissue-specific manner. The TG4 mouse strain was established using a 40-kilobase fragment containing 4.5 kilobases of 5\u27 and 27 kilobases of 3\u27 flanking sequence. The TG18 strain was developed using a 10.6-kilobase SacII fragment from the 40-kilobase fragment; this fragment contained 1.6 kilobases of 5\u27 sequence and 1.9 kilobases of 3\u27 flanking sequence. Both strains showed tissue specificity of expression of the MUC1 gene, which was very similar to the profile of expression seen in human tissues. The antibody SM-3 is directed to a core protein epitope, which is selectively exposed in breast cancers and which shows a more restricted distribution on normal human tissues. It was established that the distribution of the SM-3 epitope of PEM in the tissues of the transgenic mice is similar to that seen in humans. The transgenic mouse strains described here should form the basis for the development of a preclinical model for the evaluation of PEM-based antigens and of antibodies directed to PEM in cancer therapy

Rotation and activity of pre-main-sequence stars

We present a study of rotation (vsini) and chromospheric activity (Halpha EW) based on an extensive set of high-resolution optical spectra obtained with MIKE on the 6.5m Magellan Clay telescope. Our targets are 74 F-M dwarfs in the young stellar associations Eta Cha, TW Hydrae, Beta Pic, and Tuc-Hor, spanning ages from 6 to 30 Myr. While the Halpha EW for most F and G stars are consistent with pure photospheric absorption, most K and M stars show chromospheric emission. By comparing Halpha EW in our sample to results in the literature, we see a clear evolutionary sequence: Chromospheric activity declines steadily from the T Tauri phase to the main sequence. Using activity as an age indicator, we find a plausible age range for the Tuc-Hor association of 10-40 Myr. Between 5 and 30 Myr, we do not see evidence for rotational braking in the total sample, thus angular momentum is conserved, in contrast to younger stars. This difference indicates a change in the rotational regulation at 5-10 Myr, possibly because disk braking cannot operate longer than typical disk lifetimes, allowing the objects to spin up. The rotation-activity relation is flat in our sample; in contrast to main-sequence stars, there is no linear correlation for slow rotators. We argue that this is because young stars generate their magnetic fields in a fundamentally different way from main-sequence stars, and not just the result of a saturated solar-type dynamo. By comparing our rotational velocities with published rotation periods for a subset of stars, we determine ages of 13 (7-20) Myr and 9 (7-17} Myr for the Eta Cha and TWA associations, respectively, consistent with previous estimates. Thus we conclude that stellar radii from evolutionary models by Baraffe et al. (1998) are in agreement with the observed radii within +-15%. (abridged)Comment: 40 pages, 8 figures, ApJ, in pres

Structure of S. aureus HPPK and discovery of a new inhibitor

The first structural and biophysical data on the folate pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), from the pathogenic bacterium Staphylococcus aureus is presented. HPPK is the second essential enzyme in the folate biosynthesis pathway, responsible for catalysing pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl- 7,8-dihydropterin, HMDP). In-silico screening led to the discovery of a substrate competitive inhibitor, San1, which was subsequently co-crystallised with HPPK. A 1.65 Å resolution x-ray structure showed this to bind at the pterin site sharing many of the key intermolecular interactions of the substrate. ITC and SPR measurements yielded an equilibrium binding constant, Kd, of ~13 μM for San1. An IC50 of ~12 μM was determined by means of a new convenient tri-enzyme-coupled spectrophotometric assay. ITC and SPR further showed that the San1 inhibitor has no requirement for magnesium or ATP cofactor for competitive binding to the substrate site. According to 15N heteronuclear NMR measurements, the fast motion of the pterin loop (L2) is partially dampened in the ternary complex between SaHPPK, HMDP and , -methylene adenosine 5-triphosphate (AMPCPP), but the ATP loop (L3) remains mobile on the μs timescale. In contrast, for the SaHPPK/San1/AMPCPP ternary complex, loop L2 becomes rigid on the fast timescale and loop L3 becomes more ordered which are supported by a large entropic penalty associated with San1 binding as revealed by ITC. Backbone assignments and chemical shift perturbations implicate the sulphur in San1 as a likely important loop L2/L3 stabilizing mediato

Acute Flares of Knee Osteoarthritis (the ACT-FLARE Study): Protocol for a Web-Based Case-Crossover Study in Community-Dwelling Adults.

BACKGROUND The cardinal feature of osteoarthritis (OA) is pain. Although heterogeneity in pain and function have been demonstrated in the long-term course of OA, the more proximate determinants of acute flare-ups remain less clear. How short-term intermittent or transient exposures trigger acute flare-ups has important implications for effective and sustainable self-management strategies. OBJECTIVE The primary objective of this study is to identify potential triggers of acute flares in knee OA. Secondary objectives are to determine their course and consequences and describe high-risk participant profiles. METHODS We carried out a Web-based case-crossover study. This study aims to recruit 620 community-dwelling adults aged ≥40 years, resident in England, and who have knee pain, with or without a recorded diagnosis of knee OA, and no preexisting diagnosis of inflammatory arthropathy. Participants will be recruited via 3 routes: (1) general practice registers, (2) offline community advertisement, and (3) online social media advertisement. By using questionnaires comparing periods before participants' self-reported flare-up episodes (hazard periods) with periods during the study when their knee OA symptoms are stable (control periods), triggers preceding flare-ups will be identified and examined using conditional logistic regression. Time-to-resolution of flare-up will be examined by monitoring people's daily pain, bothersomeness, and medication usage until the participant reports when their flare-up episode ends. Rates of flare-ups will be examined across different participant and flare characteristics using regression models to identify high-risk participant profiles. A study-specific Patient Advisory Group (PAG) is providing suggestion, input, and ongoing support for all stages of the research process. RESULTS Participant recruitment opened in July 2018 and is anticipated to continue for 6 months. The study results will be disseminated through a number of channels, including relevant national or international conferences and peer-reviewed publication in a medical journal, via advocacy or charity organizations, such as Versus Arthritis and across social media. Findings will be fed back to members of our PAG, study participants, and clinicians from participating primary care general practices. The PAG will also take an active role in the overall dissemination strategy. CONCLUSIONS This study will provide empirical evidence to help patients identify common knee OA flare triggers and provide health care professionals with questions to identify patients at most risk of frequent flare-ups. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/13428

The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma

Health service utilization patterns of primary care patients with osteoarthritis

Contains fulltext : 53455.pdf ( ) (Open Access)BACKGROUND: To assess factors associated with visits to GPs, orthopaedists, and non-physician practitioners of complementary medicine (alternative practitioners) by primary care patients with osteoarthritis (OA). METHODS: Cross-sectional survey among 1250 consecutively addressed patients from 75 primary care practices in Germany. All patients suffered from OA of the knee or hip according to ACR criteria. They received questionnaires collecting sociodemographic data, data about health service utilisation, prescriptions, comorbidities. They also included established instruments as the Arthritis Impact Measurement Scale (AIMS2-SF) to assess disease-specific quality of life and the Patient Health Questionnaire (PHQ-9) to assess depression. Hierarchical stepwise multiple linear regression models were used to reveal significant factors influencing health service utilization. RESULTS: 1021 of 1250 (81.6%) questionnaires were returned. Nonrespondents did not differ from participants. Factors associated with health service use (HSU) varied between providers of care. Not being in a partnership, achieving a high score on the PHQ-9, increased pain severity reflected in the "symptom" scale of the AIMS2-SF, and an increased number of drug prescriptions predicted a high frequency of GP visits. The PHQ-9 score was also a predictor for visits to orthopaedists, as were previous GP contacts, a high score in the "symptom" scale as well as a high score in the "lower limb scale" of the AIMS2-SF. Regarding visits to alternative practitioners, a high score in the AIMS -"social" scale was a positive predictor as older people were less likely to visit them. CONCLUSION: Our results emphasize the need for awareness of psychological factors contributing to the use of health care providers. Addressing the revealed factors associated with HSU appropriately may lead to decreased health care utilization. But further research is needed to assess how this can be done successfully